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«1 Introduction.............................................................. 2 2 ...»

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Therapeutic Control of Hepatitis C Virus:

The Role of Neutralizing Monoclonal


Z.Y. Keck, K. Machida, M.M.C. Lai, J.K. Ball, A.H. Patel,

and S.K.H. Foung(*)


1 Introduction.............................................................. 2

2 Evolutionary Dynamics of HCV Envelope Genes................................. 3 3 Humoral Immunity......................................................... 6

3.1 Human Antibodies to Conformational Epitopes............................. 8

3.2 Virus Neutralization with HCV Retroviral Pseudoparticles..................... 10

3.3 Virus Neutralization with Cell Culture Infectious HCV Virions................. 11

3.4 Virus Neutralization Potency with HCV Virions............................. 12

3.5 Antibodies to Linear Epitopes........................................... 13 4 Mechanisms of Virus Neutralization........................................... 17 5 Mechanisms of Immune Escape.............................................. 18

5.1 Mutational Escape from Cellular Immunity................................. 18

5.2 HCV Infection of B Cells Induces Ig Hypermutation, Altering B Cell Immunity...................................................... 22

5.3 Antibody-Dependent Enhancement of Infection............................. 25

5.4 Potential Roles of Lipoproteins and Glycans to Modify Antibody Binding to Epitopes Mediating Virus Neutralization......................... 25 6 Final Remarks............................................................ 26 References.................................................................. 27


Liver failure associated with hepatitis C virus (HCV) accounts for a substantial portion of liver transplantation. Although current therapy helps some patients with chronic HCV infection, adverse side effects and a high relapse rate are major problems. These problems are compounded in liver transplant recipients as reinfection occurs shortly after transplantation. One approach to control reinfection is the combined use of specific antivirals together with HCV-specific antibodies.

Indeed, a number of human and mouse monoclonal antibodies to conformational and linear epitopes on HCV envelope proteins are potential candidates, since they have high virus neutralization potency and are directed to epitopes conserved across S.K.H. Foung Department of Pathology, Stanford Medical School Blood Center, 3373 Hillview Avenue, Palo Alto, CA, 94304, USA e-mail: sfoung@leland.stanford.edu

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diverse HCV genotypes. However, a greater understanding of the factors contributing to virus escape and the role of lipoproteins in masking virion surface domains involved in virus entry will be required to help define those protective determinants most likely to give broad protection. An approach to immune escape is potentially caused by viral infection of immune cells leading to the induction hypermutation of the immunoglobulin gene in B cells. These effects may contribute to HCV persistence and B cell lymphoproliferative diseases.

1 Introduction

Over 170 million people worldwide are infected with hepatitis C virus (HCV). A large proportion of these individuals will harbor a chronic infection that eventually leads to the development of severe liver disease, liver failure, and hepatocellular carcinoma (Major et al. 2001). In the United States, 40% of patients needing liver transplantation have HCV-associated diseases. While current therapy with pegylated interferon and ribavirin is effective in some patients with chronic HCV hepatitis, adverse side effects and a high relapse rate, particularly in individuals infected with genotype 1 virus, are major problems. These problems are compounded in liver transplant recipients, where HCV reinfection occurs shortly after transplantation and antiviral treatment can only start at a later time point (Biggins and Terrault 2005). Allograft failure due to HCV reinfection is the most common cause of retransplantation and death (Charlton et al. 1998). HCV reinfection occurs immediately after transplantation and viral replication starts within hours of surgery (Garcia-Retortillo et al. 2002). Risk factors associated with recurrent and severe liver disease include donor and recipient age, acute rejection, human cytomegalovirus infection, and higher levels of HCV viral load. Serum viral load increases rapidly and peaks by 4 months after transplantation with titers up to 20 times higher than pretransplant levels at 1 year after transplantation (Everhart et al. 1999). Treatment of acute rejection with steroids leads to further increases in viral load. Combined treatment with pegylated interferon and ribavirin is more effective than interferon alone (Biggins and Terrault 2005), but combined therapy is associated with more than 50% of patients needing to stop treatment because of ribavirin-associated anemia. The occurrence of HCV reinfection in liver transplant recipients indicates that the prior immunity in HCV patients does not provide protection. Clearly, more effective strategies are needed for treating and preventing HCV reinfection among liver transplant recipients, and more understanding on the mechanisms of HCV immune escape is needed.

One possibility to control posttransplantation HCV reinfection is the combined use of specific antivirals together with HCV-specific antibodies. This type of approach is widely used for the control of hepatitis B virus (HBV) after liver transplantation, where the standard of care is pretransplant treatment with lamivudine for at least 4 weeks followed by posttransplant treatment with combined lamivudine and HBV immunoglobulin (Schreibman and Schiff 2006). When lamivudine or other Therapeutic Control of Hepatitis C Virus 3 nucleoside analogs are used alone, emergence of HBV escape mutants is a major concern (Perrillo et al. 2001; Fung et al. 2006). Similarly for HCV, a number of small molecule inhibitors of HCV NS3-4A protease and NS5B polymerase are in advanced stage clinical studies, potentially providing new therapeutic options (reviewed in De Francesco and Migliaccio 2005). However, a growing concern is that a high and errorprone viral replication will eventually lead to resistance to these antivirals. Our proposal is that virus neutralizing (Vn) antibodies when used in combination with antivirals could achieve the needed therapeutic outcome and minimize escape virus mutants.

Selected antibodies ideally should be broadly reactive to different HCV genotypes, each inhibiting at different steps of virus entry, and be synergistic in their ability to control virus infection. At least two antibodies to different epitopes are proposed to minimize the concern of escape mutants. We and others have shown that the majority of HCV antibodies with the broadest and most potent Vn activities recognize conformational epitopes (Habersetzer et al. 1998; Allander et al. 2000; Ishii et al. 1998).

Nonetheless, Vn monoclonal antibodies (MAbs) to conserved linear epitopes have been identified and should be considered as therapeutic candidates (Owsianka et al.

2001, 2005; Tarr et al. 2006). While antibodies offer an as yet under-explored avenue for HCV treatment, there are a number of factors that will influence the efficacy of this approach and therefore dictate the most appropriate strategy. These factors are considered below.

2 Evolutionary Dynamics of HCV Envelope Genes

HCV can be classified into six genetically distinct genotypes and further subdivided into a large number of subtypes, of which the six major genotypes differ by approximately 30% and the subtypes differ by 20%–25% at the nucleotide level (Simmonds et al. 2005). A significant challenge for vaccine and immunotherapeutic development is the identification of protective epitopes conserved in the majority of viral genotypes and subtypes. This problem is compounded by the fact that the envelope E1E2 proteins, the natural targets for the Vn response, are two of the most variable proteins (Bukh et al. 1995). The error-prone nature of the RNAdependent RNA polymerase together with the high HCV replicative rate in vivo (Neumann et al. 1998) results in the production of viral quasispecies (Martell et al.

1994; Bukh et al. 1995). Quasispecies can respond to and overcome a variety of selective pressures including host immunity (Cooreman and Schoondermark-Van de Ven 1996; Cooreman and Schoondermark-Van de Ven 1996); chronic infection arises, at least in part, through the outgrowth of immune escape mutants (Frasca et al. 1999; Farci et al. 2000; Majid et al. 1999; Ray et al. 1999; Sullivan et al. 1999).

The envelope glycoprotein genes display some of the highest levels of genetic heterogeneity, with E2 exhibiting greater variability at the quasispecies level than E1.

Mean pair-wise genetic distances, calculated for translated HCV protein sequences spanning the entire E1E2 coding region, highlight regions of high and low variability and are distributed throughout all of the E1E2 genes (Fig. 1).

4 Z.Y. Keck et al.

Fig. 1 Mean pair-wise distance plot for E1E2 amino acids and synonymous sites generated for HCV sequences representative of genotypes 1 through 6 (98 isolates, 543 codons). Mean p-distance at specific amino acid positions are represented by black bars in the foreground.

Mean p-distance at synonymous sites in homologous codons are represented by gray bars in the background. Positions are relative to homologous codons in the H77 reference strain polyprotein.

Dotted columns represent HVR1 and HVR2. The labeled black bars located above the diversity plot represent the relative positions of characterized Vn and non-Vn MAbs recognizing linear epitopes While high levels of amino acid diversity are evident, pair-wise distances calculated at synonymous (silent) sites revealed that a considerable proportion of genetic variation across HCV genotypes 1–6 is due to synonymous substitution. These high levels of synonymous site diversity observed throughout E1 and E2 indicate these genes are under strong purifying selection. Much of the current knowledge of adaptive evolution within the envelope genes during HCV chronic infection is based on averaged estimates of synonymous (dS) and nonsynonymous (dN) nucleotide substitution rates across very small regions of the envelope genes such as the first hypervariable region (HVR1; Curran et al. 2002; Gretch et al. 1996; Honda et al.

1994; McAllister et al. 1998; Smith 1999). However, these averaging types of analysis are too blunt to dissect out exact evolutionary mechanisms leading to antibody escape. Averaged dN/dS ratios across regions are highly conservative, as only a few codons within the protein may be under diversifying selection. The signal is therefore diluted in an overbearing purifying selective background that arises through strong functional constraint. To overcome analytical problems associated with differential selection across a region, the distribution of the dN/dS ratio (w) can now be estimated for individual amino acids by assessing competing models of codon substitution within a maximum likelihood (ML) framework (Yang and Bielawski 2000).

These ML methods have recently been applied to the identification of site-specific adaptive mutations in human immunodeficiency virus (HIV) env genes (Choisy et al. 2004) and partial E1E2 sequence data sets from individuals with chronic (Brown et al. 2005) and acute phases of HCV infection (Sheridan et al. 2004). The latter study extended earlier findings (Puig et al. 2004; Farci et al. 2000) revealing a statistically significant association between outcome of acute phase infection and the number of positively selected sites (Sheridan et al. 2004). Importantly, these methods can help pinpoint those residues and regions that the virus can easily change to Therapeutic Control of Hepatitis C Virus 5 help evade both cellular and humoral responses as well as those that are under strong functional constraint. This information is key to devising appropriate immunotherapeutic approaches, since utilizing antibodies that target highly conserved functionally constrained regions will minimize the likelihood of antibody escape and maximize breadth of action and therapeutic potential.

Analysis of viral evolution has shown that the amino terminus of the E2 envelope protein contains residues that have a very high propensity for adaptive change.

Indeed, this is the only locus within E1E2 that harbors positively selected sites that are common across all genotypes (Fig. 2). This region, known as the first hypervariable region (HVR1), is the major determinant for strain-specific Vn antibody responses (Farci et al. 1994, 1996; Bartosch et al. 2003a; Rosa et al. 1996; Shimizu et al. 1994). There is increasing evidence that acute infection outcome is correlated to the rate and nature of nucleotide substitutions within the envelope genes (Farci et al. 2000; Ray et al. 1999; Sheridan et al. 2004). Patients harboring stable HVR1 quasispecies frequently resolve infection, while those with evidence of a rapidly evolving quasispecies develop chronic infection (Farci et al. 2000; Ray et al. 1999).

Evolution of the viral quasispecies continues during the chronic phase (Brown et al.

2005; Gretch et al. 1996), and differences in evolutionary rates and disease severity in individuals with differing levels of immunocompetency highlight the importance of antibody responses in controlling the infection (Booth et al. 1998; Kumar et al.

1994). While there is strong evidence that antibodies directed to this region correlate with a beneficial outcome, the limited cross-reactivity of most HVR1-specific responses presents a serious problem to their wider therapeutic potential. A recent H77 ETHVTGGSAGRTTAGLVGLLTPGAKQNIQLINT#GSWHI# 423

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